Glomerular disease is one of the major causes of chronic renal failure. During the past five years, it has been suggested that various molecules such as cytokines/growth factors and proteolytic enzymes may be involved in the pathogenesis of glomerular injury as well as in the induction of proteinuria. M. Kashgarian and R. B. Sterzel, Kidney Int. 41, 524 (1992); W. H. Baricos and S. V. Shah, Kidney Int. 40, 161 (1991). However, many recent studies have used cultured cells or affected tissue, and therefore do not generate an understanding of the pathological role of such molecules in the generation of injury in vivo. An important challenge in this field is to identify molecular mediators pivotal in different types of glomerular damage, for example, through use of an appropriate in vivo system to select candidate molecules. Currently, there are no such methods appropriate for this purpose. Thus, it would be useful to establish methods suitable for assessing the pathophysiological function of specific molecules in situ, i.e., within renal glomeruli, using gene transfer technology.
The most commonly used techniques for delivering exogenous nucleic acid into cells involve the use of viral vectors. These vectors are advantageous in that they can infect large percentages of recipient cells and can integrate into the cell genome. The vital vectors are often constructed to be replication-defective once they have transfected a cell line. Other viral vectors that have been proposed or used for delivering nucleic acid into cells include adenovirus, adeno-associated virus, herpes virus and poliovirus vectors. The retroviral and adeno-associated virus vectors are most often proposed or used for ex vivo gene therapy, i.e., delivery of an exogenous DNA construct into cells temporarily removed from the body of the patient.
Hereinafter, exogenous nucleic acid construct or exogenous gene construct refers to a nucleic acid sequence originating outside a recipient cell and introduced into a recipient cell by a nucleic acid delivery technique. A nucleic acid or gene construct may be manufactured using recombinant DNA technology known in the art, or may be a nucleic acid fragment purified from a source material without further manipulation. The exogenous gene may be entirely composed of homologous sequences, i.e., sequences cloned, isolated, or derived from the same species from which the recipient cells derive. Alternatively, all or a portion of the exogenous gene may be composed of sequences from species other than the species from which the recipient cells derive, hereinafter termed heterologous sequences. The exogenous gene construct may be natural in that none of the regulatory sequences and coding sequences that may be a part of the gene are substantially or intentionally altered, or the exogenous gene construct may be chimeric in that sequence fragments from various sources are present in the final gene construct. Examples of exogenous nucleic acid constructs introduced into cells include constructs expressing bacterial proteins, oncogenes, cell surface molecules, and antisense sequences. Minoru, S., et al., EMBO J. 9:2835 (1990); Gossett, L., J. Cell Biol. 106:2127 (1988); Townsend, S. and Alison, P., Science 259:368 (1993); Trojan, J., et al., Science 259:94 (1993).
Gene transfer has been effected into various organs including bone marrow, skin, brain, heart, muscle, lung, liver, kidney, and arterial wall. J. W. Larrick and K. L. Burck, Gene Therapy: Application of Molecular Biology (Elsevier, New York 1991) chap. 5-7; H. Lin, et al. Circulation 82, 2217 (1990); R. J. Bosch, A. S. Woolf, L. G. Fine, Exp. Nephrol. 1, 49 (1993). In these cases, exogenous genes have been applied to the target organ or tissue by direct injection or local instillation of materials. In the kidney, however, the glomeruli are small structures (100-200 .mu.m in diameter) scattered throughout the renal cortex (3.times.10.sup.4 -1.times.10.sup.6 glomeruli/kidney) and, therefore, cannot be targeted by conventional approaches. Direct injection of viral vectors or DNA-liposome complexes into the renal circulation potentially could cause other renal cell types as well as other organs to be exposed to the exogenous DNA.
Woolf et al., Kidney Int. 43 (Suppl. 39): S116-S119 (1993) disclosed two approaches to gene therapy of the kidney. The first approach involved transplantation of embryonic metanephric tissue that had been transduced with a reporter gene carried by a retrovital vector. In contrast to adult tissue, the embryonic metanephros contains mitotically active cells, required for integration and expression of the retrovital vector. Pieces of the transduced embryonic tissue were transplanted under the renal capsule of adult mice or into the renal cortex of neonatal mice. The authors admitted that long-term survival of the metanephric transplants was limited by ischemia and immune rejection. This approach is dependent on a source of compatible embryonic tissue, and requires surgical intervention in the patient's kidney.
The second approach involved direct injection of retrovirus vectors into kidneys of adult mice. A small number of proximal tubular cells were found to express a reporter gene a few days after injection of retrovirus. Such direct administration of virus creates the possibility for non-kidney tissues and organs to be exposed to the vector. Moreover, since retroviruses require dividing cells for integration and long-term expression, Woolf et al. needed to create a proliferative environment in the adult kidney. They accomplished this by treating the recipient mice with folic acid in order to create generalized and sub-acute damage to the kidney. This, in turn, generated a round of repair proliferation that facilitated integration of the retrovirus vectors. Woolf et al. pointed out that this approach "clearly . . . would be unacceptable if gene transfer into human kidneys was contemplated, unless the injury phase could be tightly controlled."